Functional/Structural Studies of Mammalian Neuraminidase

描述(由申请人提供)： 这项建议的范围是研究哺乳动物溶酶体神经氨酸酶(NEU1)在正常细胞代谢中的功能，以及它的缺失在人类疾病中的后果。NEU1属于广泛存在的唾液酸酶超家族。哺乳动物神经氨酸酶包括胞浆、溶酶体和质膜异构体，关于这些水解酶，特别是NEU1的生理作用的线索直到最近才出现。NEU1通过去除末端唾液酸残基启动唾液酸糖结合物的水解。该酶在唾液酸酶中是独一无二的，因为它必须与保护性蛋白/组织蛋白A(PPCA)相关联，以实现细胞内的路由和溶酶体的激活。最后，NEU1与两种神经退行性代谢性疾病有关：唾液症是由NEU1的结构损害引起的，而半乳糖化病(GS)是由于缺乏PPCA而引起的NEU1和(3-半乳糖苷酶)的联合缺陷。拟议的研究基于三个具体目标。在目标1中，我们将研究NEU1和PPCA之间的结构-功能关系。PPCA前体的3D结构将被用于靶向突变这两种蛋白和在涎腺病患者中发现的NEU1突变之间的潜在接触位点。我们还将使用跨越全长PPCA和NEU1的重叠多肽来确定对NEU1/PPCA相互作用、细胞内转运和激活至关重要的结构域。这些生化研究将结合起来确定Neu1/PPCA复合体的3D结构。在目标2中，我们将比较Neu1~*~小鼠和PPCA^~~小鼠的特征，以确定涎腺病和GS的分子基础，并鉴定尚不清楚的Neu1在正常细胞生理中的功能。在目标3中，我们将在两个模型中实施各种酶替代治疗方法，以评估这些疾病的系统表型的纠正。我们有能力开发这一系列研究，因为我们已经为拟议的研究建立了适当的遗传和生化系统，并且可以依靠杰出的结构生物学家的专业知识来完成该项目的结晶学部分。我们的总体目标是更广泛地了解NEU1在正常生理学和神经退行性疾病、涎症和GS的病理生理学中的作用。这些疾病主要影响婴儿和儿童。这些拟议研究的结果将增加我们对NEU1功能的了解，并改进未来治疗这些灾难性疾病的儿科患者的设计。

DESCRIPTION (provided by applicant): The scope of this proposal is to study the function of mammalian lysosomal neuraminidase (NEU1) in normal cell metabolism, and the consequences of its loss in human diseases. NEU1 belongs to the ubiquitous superfamily of sialidases. Mammalian neuraminidases include cytosolic, lysosomal, and plasma membrane isoforms, and clues about the physiologic roles of these hydrolases, in particular NEU1, have emerged only recently. NEU1 initiates the hydrolysis of sialo-glyconjugates by removing terminal sialic acid residues. The enzyme is unique among sialidases in that it must be associated with protective protein/cathepsin A (PPCA) for intracellular routing and lysosomal activation. Finally, NEU1 is linked to 2 neurodegenerative diseases of metabolism: Sialidosis is caused by structural lesions in NEU1, and galactosialidosis (GS), a combined deficiency of NEU1 and (3-galactosidase, is caused by the absence of PPCA. The proposed studies are based on 3 Specific Aims. In Aim 1, we will investigate the structure- function relationship between NEU1 and PPCA. The 3D structure of the PPCA precursor will be used to target mutagenesis of potential contact sites between the 2 proteins and NEU1 mutations identified in patients with sialidosis. We will also use overlapping peptides that span the full-length PPCA and NEU1 to identify domains crucial for NEU1/PPCA interaction, intracellular transport, and activation. These biochemical studies will be coupled to determine the 3D structure of the Neu1/PPCA complex. In Aim 2, we will compare the characteristics of Neu1~*~ mice and PPCA^~ mice to determine the molecular bases of sialidosis and GS and to identify yet unknown Neu1 functions in normal cell physiology. In Aim 3, we will implement various enzyme replacement therapy approaches in both models to assess the correction of the systemic phenotypes in these diseases. We are in position to develop this line of investigation, because we have established appropriate genetic and biochemical systems for the proposed studies and can rely on the expertise of an outstanding structural biologist for the crystallography part of the project. Our overall goal is to gain a broader understanding of NEU1 function in normal physiology and in the pathophysiology of the neurodegenerative diseases sialidosis and GS. These diseases affect primarily infants and children. Findings from these proposed studies should increase our knowledge about NEU1 function and improve the design of future therapies for pediatric patients with these catastrophic diseases.